Determination of the content of p-methoxy cinnamic acid ethyl ester in hawthorn - Master's thesis - Dissertation

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Abstract: This study aims to develop a reliable method for the determination of ethyl p-methoxycinnamate in hawthorn. The content of p-methoxy cinnamate was analyzed using gas chromatography (GC). A SE-30 stationary phase column was used with a column temperature of 20°C. The results showed a strong linear relationship (r = 0.9995), an average recovery rate of 97.8%, and a relative standard deviation (RSD) of 1.1% (n = 5). This method offers an effective way to assess the quality of medicinal hawthorn materials.

Keywords: Gas chromatography; Hawthorn; Ethyl p-methoxycinnamate

Hawthorn, scientifically known as Kaempferia galanga L., is a plant from the Zingiberaceae family. It is commonly cultivated in regions such as Taiwan, Guangdong, Guangxi, and Yunnan. The dried rhizomes are traditionally used for their warming, digestive, and pain-relieving properties. According to the 2000 edition of the pharmacopoeia, the volatile oil content should not be less than 4.5% (ml/g), with ethyl p-methoxy-cinnamate being the main component. This paper presents a GC-based method for determining the ethyl p-methoxycinnamate content in hawthorn, which is simple and suitable for quality control purposes.

1. Instruments and Reagents

1.1 Instrument: Puri GC7800 gas chromatograph, ultrasonic cleaner.

1.2 Reagents: The reference substance of ethyl p-methoxycinnamate was provided by the China National Institute for the Control of Pharmaceutical and Biological Products, with batch number 0835.9601. It was purified by recrystallization and had a purity of over 98% after GC analysis. All other reagents were of analytical grade.

1.3 Sample Preparation: Commercially available hawthorn samples were used. The roots of Kaempferia galanga L. were identified by our laboratory. The samples were ground and sieved through a 40-mesh sieve before testing.

2. Methods and Results

2.1 Chromatographic Conditions: A glass column (2.1 m long, 0.3 cm in diameter) was packed with SE-30 as the stationary phase at a coating concentration of 10%. The column temperature was set at 200°C, while the inlet and detector temperatures were both 250°C. The carrier gas flow rate was 80 kPa, hydrogen flow rate was 100 kPa, air flow rate was 50 kPa, and the sensitivity was set to 10⁻¹.

2.2 Extraction Conditions: Methanol and ethyl acetate were tested as solvents. Ultrasonic extraction for 30 minutes yielded the highest extraction efficiency. The extraction time was optimized, and complete extraction was achieved within 30 minutes.

2.3 Linear Relationship: A series of reference solutions with concentrations of 0.5, 1.0, 2.0, 5.0, and 10.0 mg/mL were prepared. Each solution was injected into the GC system, and the peak areas were recorded. A calibration curve was plotted with injection amount on the x-axis and peak area on the y-axis. The regression equation was Y = 316412.8X - 43265.8, with r = 0.9995, indicating good linearity in the range of 1–20 μg.

2.4 Precision and Stability: Five consecutive injections of the same sample showed an RSD of 0.8% based on peak area. The stability test revealed that the samples remained stable for up to 24 hours, with an RSD of 1.0%.

2.5 Reproducibility: Five portions of the same sample were tested according to the procedure outlined in section 2.7. The measured contents were 39.3, 39.6, 40.9, 40.2, and 40.3 mg/g, with an average of 40.1 mg/g and an RSD of 1.6%.

2.6 Recovery Test: Accurately weighed hawthorn samples (0.5 g) with known ethyl p-methoxycinnamate content were spiked with a known amount of reference substance. The recovery rate was calculated as 97.8% with an RSD of 1.1% (n = 5).

2.7 Sample Analysis: Approximately 1 g of hawthorn was placed in a flask, and 10 mL of methanol was added. The weight was recorded, followed by sonication for 30 minutes. The weight loss was compensated, and the solution was filtered. The filtrate served as the test solution. Both the reference solution (4.0 mg/mL) and the test solution were injected into the GC system, and the results are summarized in the table.

3. Discussion

The quality of hawthorn is often judged by its aromatic scent and spicy taste. The volatile oil content typically ranges between 3% and 4%, with ethyl p-methyl cinnamate as the primary component. Determining the ethyl p-methoxycinnamate content provides a reliable means of controlling the quality of medicinal hawthorn. Additionally, this method serves as a reference for improving the quality standards of hawthorn in the future.